The goal of pharmaceutical chemistry has always been to identify chemical compounds that have the ability to affect specific biological pathways or reactions so that they are useful as therapeutic agents or as probes of biological activity. Recent developments in synthetic chemistry have greatly expanded scientists""ability to rapidly produce potentially interesting chemical compounds, and to assay their biological and chemical activities. Most notably, the burgeoning field of combinatorial chemistry provides techniques for the rapid and facile generation of large numbers of compounds (see, e.g., xe2x80x9cCombinatorial Chemistryxe2x80x9d, Chem. and Eng. News, Feb. 24, 1997, p. 43; Thompson, L. A., Ellman, J. A., Chem. Rev. 1996, 96, 555).
Combinatorial methods are available for use in the solution phase, the solid phase, or combinations thereof. One particularly powerful solid-phase technique, known as xe2x80x9csplit and poolxe2x80x9d synthesis, allows large numbers of compounds to be produced, each of which is separately attached to its own solid support (Furka, A. et al., Int. J. Pept. Protein Res. 1991, 37, 487-493). However, one complication of this method is that, because the solid supports are recovered as mixed pools, either deconvoluting or encoding strategies are required to determine the chemical structure of compounds with desired activities.
A large variety of different deconvolution and encoding techniques have been developed to facilitate the analysis of chemical compounds produced by split-and-pool techniques (See, e.g., Czarnik, A. W., Curr. Op. Chem. Biol., 1997, 1, 60). One of the earliest encoding methods employed oligonucleotide tags for identification of libraries of random oligomers (WO 93/06121). Although an advantage of this method is that tag analysis can be readily accomplished through polymerase chain reaction amplification (PCR), the tags are not sufficiently stable to survive the synthetic conditions required for production of small molecule libraries.
Other available encoding schemes include the use of fluorophenyl ether tags (Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 1993, 90, 10922; Nestler et al., J. Org. Chem. 1994, 59, 4723). These tags have the advantage that they are resistant to most of the reaction conditions used for organic synthesis. However, the tags are analyzed by gas chromatography and electron capture, and therefore cannot be assayed without first being cleaved from the solid support. Difficulties are often encountered in attempts to analyze the small quantities of tag that are released in these methods.
There has been one report of a tag compound, 3, 5-dimethoxy benzoic acid labeled with 13C, being analyzed directly on a solid support (a Wang resin), without first being released by cleavage (J. Am. Chem. Soc. 1996, 118, 2305; WO 97/14814). However, the method described lacked sensitivity, and required time-intensive 13C nuclear magnetic resonance (NMR) spectroscopy techniques. Unfortunately, the technique therefore cannot be practically applied to the rapid analysis of large numbers of compounds.
Therefore, there remains a need to develop an improved system for encoding reaction history or direct structural information about chemical compounds synthesized on the solid phase. Preferably, the system should allow on-bead analysis, and should employ chemically robust detectable tags.
The present invention provides an improved system for encoding the reaction history and/or molecular structure of chemical compounds attached to a solid support. In general, the invention provides an identification unit comprising a binding pair in which a first ligand, component one, is associated with the solid phase, and a second ligand, component two, is employed to specifically recognize the first ligand. Component one need not be separable from the solid support. Component one is sometimes referred to herein as the xe2x80x9ctagxe2x80x9d.
In certain preferred embodiments of the invention, component one is attached to the solid phase during the synthesis of the chemical compound. In such circumstances, component one preferably designates a particular reaction step so that the synthetic history of the chemical compound is recorded in a series of component ones attached to the solid support. Each component one is then preferably detected by means of its component two binding partner, after the synthesis is complete.
Component two may be any ligand capable, of specific interaction with a component one. Component two need not be chemically robust, as it is not present during the synthesis reactions. However, component two must be selected so that its interaction with component one is detectable. Any method of detection is sufficient. For example, component two may include (i.e., may be covalently linked to) or be otherwise associated with (i.e., by other than covalent linkage) a fluorescent, luminescent, or radioactive moiety. In certain preferred embodiments, component two is associated with a detectable moiety such as a nucleic acid molecule (having a selected nucleotide sequence defining a particular component one-component two interaction) whose signal is subject to amplification (e.g., by PCR), so that the detectable moiety can be identified even when present at very low levels. The steps involved in detecting this binding interaction preferably include, (1) providing a solid phase, a chemical compound and a tag, (2) contacting the tag with the binding partner, and (3) detecting a detectable moiety, which detectable moiety may be any moiety capable of being identified.
The present invention also provides methods of identifying chemical compounds attached to a support by (1) providing a solid support to which a chemical compound whose structure is to be determined is attached, along with a tag selected to represent a structural or synthetic feature of the chemical compound; (2) contacting the solid support with a binding partner that binds specifically and detectably to the tag; and (3) detecting binding of the binding partner to the tag, the existence of such binding being indicative of the presence of the tag on the solid support, which presence is in turn indicative of the existence of the structural or synthetic feature of the chemical compound. Preferably, (1) the step of providing involves providing a solid support comprising a plurality of attached tags, each of which is selected to represent a particular structural or synthetic feature of the chemical compound; (2) the step of contacting comprises (a) providing a plurality of binding partners, each of which specifically and detectably binds to one tag; and (b) contacting the solid support with each of the binding partners (simultaneously or sequentially); and (c) the step of detecting comprises detecting each binding partner/tag binding interaction, and thereby determining the existence of the structural or synthetic features of the compound.
Finally, the present invention provides methods for analyzing the tags off the bead. Specifically, the present invention provides techniques for creating spatially encoded split and pool libraries using the tags and binding partners described herein. According to this aspect of the present invention, (1) the beads, with attached compounds and tags, are distributed into microtiter plates at one bead per well; (2) the tags are detached from the beads by a specific releasing chemistry; (3) the tags are arrayed onto a slide; and (4) the compound represented by the tags are identified by the steps comprising (a) providing a plurality of binding partners; (b) contacting the tags arrayed on each slide with each of the binding partners; and (c) detecting each binding partner/tag binding interaction.
In another aspect, the present invention provides a kit comprising (1) a collection of chemically robust tag components capable of attachment to a solid support, and (2) a corresponding collection of detectable binding partners, each of which interacts selectively with one tag component.
xe2x80x9cEncoded combinatorial libraryxe2x80x9d An encoded combinatorial library, as that phrase is used herein, is a collection of chemical compounds where each compound is attached to a solid support that also contains information revealing the structure of the compound, either directly or by recording the reaction history that produced the compound.
xe2x80x9cTagxe2x80x9d: As used herein, the term xe2x80x9ctagxe2x80x9d means a chemical moiety, preferably a robust, small molecule that is capable of being detected by a binding partner moiety when present at less than or equal to 10xe2x88x9212 moles on the bead. This property renders the tag detectable and optionally may provide the property of rendering the tag identifiable while attached to a solid support.
xe2x80x9cChemically robustxe2x80x9d: The term xe2x80x9cchemically robustxe2x80x9d, as used herein, in reference to a tag, means that the tag can withstand the chemical reactions utilized in the synthesis of combinatorial libraries.
xe2x80x9cSmall moleculexe2x80x9d: As used herein, the term xe2x80x9csmall moleculexe2x80x9d refers to an organic compound either synthesized in the laboratory or found in nature. Typically, a small molecule is any organic molecule that can preferably be recognized by a macromolecule, and has a molecular weight of less than 1500. For example, the small molecule could be a hapten.
xe2x80x9cBinding partnerxe2x80x9d: As used herein, the term xe2x80x9cbinding partnerxe2x80x9d, xe2x80x9cbinding partner moietyxe2x80x9d, xe2x80x9cpartnerxe2x80x9d, or xe2x80x9cpartner moleculesxe2x80x9d refers to a compound or compounds capable of selectively and specifically associating with the tag component as a means to identify the tag component. A binding partner can be any compound that interacts specifically with the tag such that together they form a binding pair or identification unit. In one non-limiting example, a binding partner may be an antibody that specifically recognizes the small molecule tag. It is preferable that the binding partner has associated with it some means of detecting the antibody.
xe2x80x9cLabelxe2x80x9d: As used herein the term xe2x80x9clabelxe2x80x9d, or xe2x80x9cdetectable moietyxe2x80x9d is any means for detecting an interaction between a tag and a binding partner, thereby identifying the presence of the tag and the existence of the structural or synthetic information that the tag represents. The label may be any means of detection that can be assayed. The label provides a xe2x80x9csignalxe2x80x9d indicating which tag is being identified. In one non-limiting example, the label may be a fluorescent label associated with a binding partner.
xe2x80x9cSignalxe2x80x9d: The term xe2x80x9csignalxe2x80x9d as used herein refers to the information readout provided by the identification unit. The readout may indicate which tag is being identified. The signal may be provided by the label. In some preferred embodiments the signal is provided by a fluorescent label or a quantum dot. In other preferred embodiments the signal is provided by a nucleic acid. In yet other preferred embodiments, the signal is provided by the binding partner itself, for example where the binding partner is an identifiable nucleic acid.
xe2x80x9cIdentification unitxe2x80x9d: The term xe2x80x9cidentification unitxe2x80x9d as used herein, refers to the binding pair comprising a tag component and a binding partner moiety that together function to encode information. In one preferred embodiment, the identification unit includes a small molecule tag and any compound capable of associating specifically and detectably with that small molecule. In another preferred embodiment the identification unit includes a small molecule tag and an antibody capable of selectively associating with that small molecule. Preferably, the tag is attached to a solid support and the binding partner, with its associated means of detection, be associated with the small molecule on the solid support. Alternatively, the tag may be cleaved from the solid support for the purpose of detection.
xe2x80x9cHandlexe2x80x9d: xe2x80x9cHandlexe2x80x9d as used herein, refers to the chemical group or linker molecule used to attach a tag molecule to a glass slide or plate for creating spatially encoded split and pool libraries. A handle can be any chemical moiety capable of attaching any tag to a slide (preferably glass) also modified for attachment. Preferably, the chemical moiety is easily attached to the tag. For example, a glass slide may be derivatized with either thiol or maleimide groups to covalently capture a tag molecule via an attached thiol group.
xe2x80x9cChoicexe2x80x9d: As used-herein the term xe2x80x9cchoicexe2x80x9d means the alternative variables for a given stage in a combinatorial synthesis, such as reactant, reagent, reaction conditions, and combinations thereof. The term xe2x80x9cstagexe2x80x9d refers to a step in the sequential synthesis of a compound or ligand; the compound or ligand being the final product of a combinatorial synthesis.
xe2x80x9cAssociated withxe2x80x9d: The term xe2x80x9cassociated withxe2x80x9d, as used herein, is defined as using any means of providing physical proximity between two compositions of interest (e.g., covalent association, hydrophobic interaction, or ionic interaction). By way of example, a tag may be associated with its binding partner.
xe2x80x9cLinkerxe2x80x9d: As used herein, the term xe2x80x9clinkerxe2x80x9d means a chemical moiety that can simultaneously attach to both a solid support and a tag, thereby xe2x80x9clinkingxe2x80x9d the tag to the support. Optionally, the linker may be cleavable so that the tag may be released from the solid support by disruption of the linker. These three properties may be embodied in a single chemical structure. A linker may be embodied in a single chemical moiety, or in a collection of chemical moieties associated with one another. In this latter case, one of the chemical moieties provides the property of rendering the linker attachable to the solid support; the second chemical moiety, provides the property of rendering the linker cleavable; and the third chemical moiety provides the property of rendering the linker attachable to the tag. Desirably the chemical structures that provide a means for tag attachment and a means for tag cleavage are one and the same.
xe2x80x9cCapxe2x80x9d or xe2x80x9cCappedxe2x80x9d: As used herein, the term xe2x80x9ccapxe2x80x9d or xe2x80x9ccapped,xe2x80x9d as used in reference to applying a tag to a reaction series, means to terminate the growing reaction series with the added tag.
xe2x80x9cSupportxe2x80x9d: The materials upon which the combinatorial syntheses of this invention are performed are referred to herein interchangeably as beads, solid surfaces, (solid) substrates, particles, (solid) supports etc. These terms are intended to include:
a) solid supports such as beads, pallets, disks, capillaries, hollow fibers, needles, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, poly-acrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,Nxe2x80x2-bis-acryloyl ethylene diamine, glass particles coated with a hydrophobic polymer, etc., i.e., a material having a rigid or semi-rigid surface; and
b) soluble supports such as low molecular weight non-cross-linked polystyrene.
xe2x80x9cSpecifically associatesxe2x80x9d: as used herein in reference to the interaction between a tag component and a binding partner means that the interaction between one tag component and a specific binding partner occurs preferably over interaction with a variety of other binding partners. For example, tag A is presented with binding partners A-Z, but only binds to binding partner K. In this example, tag A xe2x80x9cspecifically associatesxe2x80x9d with binding partner K.